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Author

Bamaprasad Dutta

Date of Issue

2014

School

School of Biological Sciences

Abstract

Chromatin, a key component of eukaryotic cells composed of DNA and proteins, plays a key regulatory role by controlling accesses to the DNA template that carry genetic instructions for all cellular functions. The chromatin-associated proteome, also called chromatome, is a dynamic system which changes in response to internal and external stimuli. Through the changes, chromatome actively modulates chromatin structure to regulate developmental biology, cell cycle, epigenetics and pathobiology such as cancer development. A handful of chromatin proteomic studies were reported as of now. However, the dynamic chromatin protein binding topology which directly regulates genetic and epigenetic biology in normal development and disease remains unknown. Most characterized genetic mutations in common cancers are linked to tumor initiation and few are related to tumor progression and metastasis. Recent studies showed that epigenetic changes play a key role in tumor malignancy. Emerging clinical and experimental evidences suggest that hypoxic tumor microenvironment might alter the epigenetic marks and induce stemness in normal cancer cells and converting them into the radio- and chemo-resistance malignant cancer cell phenotypes. In addition, recent evidences also revealed the correlation between hypoxia and cell cycle regulation. Thus, characterization of the chromatome dynamics to elucidate chromatin biology by quantitative proteomic approach will provide molecular mechanistic insight of hypoxic microenvironment induced tumor malignancy. Accordingly, our aims are 1) to develop a proteomic method to study the dynamic chromatome by nuclease digestion to release proteins bound to specific chromatin region coupled with quantitative proteomic profiling; and 2) to elucidate the dynamics of chromatin biology during cell cycle and hypoxia induced tumor malignant progression by using our proteomic method developed in aim 1. In order to establish a suitable proteomic approach to study chromatome dynamics, we used rat liver chromatin as model. We used partial MNase and DNase I digestion together with iTRAQ-based quantitative proteomics to study euchromatin- and heterochromatin-associated proteins in liver cells. A total of 694 chromatin-associated proteins was identified at a high level of confidence that allow us to determine their dynamic interactions with chromatin to infer their euchromatin and heterochromatin associations and their roles in chromatin biology. We reported several novel chromatin-associated proteins and also validated the localization of Gnl3, Ncl, and Phb proteins by immunofluorescence microscopy analysis. After having established the method, we applied this partial nuclease digestion coupled with iTRAQ-based quantitative proteomic approach to study the dynamic changes of the chromatomes during cell cycle progression and hypoxia microenvironment induced cancer progression. A total of 481 and 1446 proteins were identified at high confidence level during the cell cycle and hypoxia stress based experiments respectively. Data mining suggested that identified proteins were involved in chromatin-dependent events including transcriptional regulation, chromatin re-organization, and DNA replication and repair, while the quantitative data of differential released chromatin-associated proteins by partial nuclease digestion revealed the temporal interactions of these proteins with euchromatin and heterochromatin during interphase progression and different hypoxia stress conditions. When combined with biochemical and functional assays, these data revealed a strikingly dynamic association of protein HP1BP3 with the chromatin during different stages of interphase and also during different oxygen tensions, and uncovered a novel regulatory role for this molecule in the maintenance of chromatin structural integrity and transcriptional regulation. We report that HP1BP3 protein maintains heterochromatin integrity during G1-S progression and determine the G1 phase duration by regulating G1/S transition, which critically influence cell proliferative capacity. Cell cycle chromatomic data together with other functional studies revealed the role of chromatin regulatory protein KDM1 in the regulation of S/G2 transition. Functional characterization of HP1BP3 during cancer progression showed that HP1BP3 mediated heterochromatinization was activated during hypoxia, which transformed the normal cancer cells into aggressive and malignant cancer cell phenotype through increasing cell survivability, cell motility, radio- and chemo-resistance, and stemness. Based on our experimental evidences and observations we suggested that HP1BP3 mediated heterochromatinization will be the potential target for new cancer therapy.